Objective: To explore the feasibility of inhibition of hTERT and androgen receptor (AR) gene expression simultaneously in LNCaP cells by single shRNA vector.
Methods: Templates DNA of both hTERT and AR siRNA were inserted into Pgenesil vector to construct a new vector Pgenesil-hTERT-AR-shRNA by RNAi-DNA vector technology. Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA vectors were transfected into prostate cancer LNCaP cells respectively. The levels of AR mRNA, apoptosis and proliferation of each cell group were determined by FQ-PCR, Annexin V method and MTT.
Results: The level of hTERT mRNA of control group cells and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.51 +/- 0.08) x 10(8), (7.32 +/- 0.43) x 10(7), (2.94 +/- 0.15) x 10(6), (4.45 +/- 0.25) x 10(7) and (3.17 +/- 0.18) x 10(6) (copies/ml) respectively. The level of AR mRNA of control cell groups and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.92 +/- 0.11) x 10(5), (6.47 +/- 0.32) x 10(5), (3.70 +/- 0.24) x 10(4), (1.22 +/- 0.06) x 10(4) and (7.21 +/- 0.41) x 10(3) (copies/ml) respectively. These data indicate that the expression of hTERT or AR gene could be significantly inhibited by Pgenesil-hTERT-shRNA or Pgenesil-AR-shRNA while Pgenesil-hTERT-AR-shRNA could simultaneously inhibit both hTERT and AR gene expression. The apoptosis rate and the inhibition rate of cell growth of Pgenesil-hTERT-AR-shRNA group were significantly higher than those of Pgenesil-hTERT-shRNA group or Pgenesil-AR-shRNA group (P < 0.05).
Conclusion: It is feasible to inhibit both hTERT and AR gene expression simultaneously by single shRNA vector. It will be a new research strategy of gene therapy for prostate cancer.